Role of natriuretic peptide clearance receptor in in vivo control of C-type natriuretic peptide.

C-type natriuretic peptide (CNP) is a newly described 22-amino acid peptide of endothelial cell origin, which has selective cardiovascular actions and is structurally related to atrial natriuretic peptide (ANP). Recent in vitro studies have demonstrated that an important regulatory pathway for the clearance of natriuretic peptides involves binding to a common clearance receptor [natriuretic peptide C receptor (NPR-C)]. Although CNP has also been identified as a ligand for NPR-C in binding assays, no studies have defined the in vivo interaction of CNP with NPR-C. CNP (10 ng.kg-1.min-1) followed by C-ANP-(4-23), a specific ligand for NPR-C blockade, was infused intravenously in two groups (both n = 7) of anesthetized dogs at two different doses (0.1 or 1.0 micrograms.kg-1.min-1) to permit calculation of total metabolic clearance rate (TMCR). C-ANP-(4-23) increased circulating CNP and reduced TMCR in both groups. Pulmonary metabolic clearance rate was negative at baseline, suggesting a net secretion of CNP across the lung, which was increased during CNP infusion and was abolished with NPR-C blockade. Renal and femoral metabolic clearance rates were positive at baseline and increased with CNP infusion. A decrease in cardiac output and cardiac filling pressures in response to CNP administration was potentiated by NPR-C blockade. We conclude that 1) circulating CNP achieved by CNP infusion is regulated by NPR-C in vivo, 2) the pulmonary circulation is a possible site of CNP secretion, 3) the renal and peripheral circulations are sites of CNP clearance, and 4) NPR-C blockade potentiates the selective cardiovascular actions of CNP.

Clearance receptor-mediated control of atrial natriuretic factor in experimental congestive heart failure.

Circulating atrial natriuretic factor (ANF) is regulated by clearance receptors (ANFR-C). C-ANF-(4-23) is a ring-deleted analogue of ANF, which binds specifically to ANFR-C. The present studies were undertaken to determine total metabolic (TMCR), pulmonary (PCR), and renal clearance rates (RCR) of ANF in a group of seven mongrel dogs in chronic congestive heart failure (CHF) in comparison with a control group (n = 6). TMCR was not altered in CHF [1,534 +/- 319 vs. control: 1,735 +/- 208 ml/min; P = not significant (NS)] in association with an elevation of circulating endogenous ANF (206 +/- 44 vs. control: 36 +/- 10 pg/ml; P < 0.01). Infusion of C-ANF-(4-23) reduced TMCR in both groups similarly (CHF: 753 +/- 134 vs. control: 972 +/- 156 ml/min; P = NS). PCR was lower in CHF (286 +/- 431 vs. 1,672 +/- 407 ml/min; P < 0.05), whereas RCR was not different (10 +/- 24 vs. control: 15 +/- 25 ml/min; P = NS). ANFR-C blockade did not facilitate urinary sodium excretion in CHF. These studies demonstrate that 1) TMCR does not contribute to elevated endogenous ANF in CHF; 2) total functional activity of the clearance receptor pathway is preserved in CHF; and 3) renal ANF metabolism and the clearance receptor pathway are not linked to the avid sodium retention and renal ANF resistance observed in chronic CHF.

Biologic role of atrial natriuretic factor clearance receptor in congestive heart failure.

Atrial natriuretic factor (ANF) is a circulating 28-amino acid peptide that functions in the regulation of sodium homeostasis and vascular tone. ANF metabolism occurs via degradation by neutral endopeptidase 24.11 and binding to the ANF clearance receptor (ANFR-C). The present study was performed on anesthetized dogs, normal (control) and with experimental congestive heart failure (CHF), and was designed to investigate the ability of an ANF ligand specific for ANFR-C [C-ANF-(4-23)] to increase plasma ANF and also to evaluate the influence of ANFR-C on regional pulmonary and renal ANF clearances. C-ANF-(4-23) increased plasma ANF in controls (51 +/- 15 to 123 +/- 39 pg/ml; P < 0.05) and further increased plasma ANF in CHF dogs (242 +/- 30 to 327 +/- 34; P < 0.05), demonstrating that ANFR-C plays a significant role in the overall metabolism and clearance of ANF, even with chronically elevated ANF. Infusion of C-ANF-(4-23) produced a marked decrease in ANF pulmonary clearance (PCLANF) in controls (1,018 +/- 405 to -286 +/- 383 ml/min; P < 0.05); however, PCLANF was not altered by the ANF ligand in CHF dogs [-137 +/- 174 to -106 +/- 226 ml/min; not significant (NS)], suggesting an occupancy of ANFR-C or a downregulation of this receptor with chronically elevated plasma ANF. ANF renal clearance (RCLANF) was not altered in either group by C-ANF-(4-23) infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

C-type natriuretic peptide inhibits growth factor-dependent DNA synthesis in smooth muscle cells.

We have examined the ability of C-type natriuretic peptide (CNP) to interact with guanylate cyclase-coupled natriuretic peptide receptors by measuring its ability to stimulate intracellular guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in cultured bovine aortic endothelial (BAE) and bovine aortic smooth muscle (BASM) cells. Our experiments indicate that CNP is unable to stimulate the production of cGMP in BAE cells, whereas both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) markedly elevate cGMP levels in these cells (ANP = BNP >> CNP). In contrast, CNP is the most effective of the three peptides with respect to the stimulation of cGMP levels in BASM cells, fetal human vascular smooth muscle cells, and rat A10 cells (CNP >> ANP > BNP), with the maximal level of stimulation being approximately 5- to 10-fold over that observed for ANP. We have also shown that CNP is able to inhibit serum- and growth factor-induced DNA synthesis in BASM cells. Low concentrations of CNP (20 x 10(-9) M) inhibit up to 80% of the [3H]-thymidine incorporation induced by basic fibroblast growth factor, platelet derived growth factor, epidermal growth factor (EGF), and heparin binding EGF-like growth factor. These data indicate that, although CNP has been detected only in the central nervous system and not in the circulation, it may possess multiple effects on vascular tissue.

Clearance receptor and neutral endopeptidase-mediated metabolism of atrial natriuretic factor.

A novel small linear C-atrial natriuretic factor receptor ligand [C-ANF-(11-15)] and phosphoramidon (PHO) were used to determine the effects of C-ANF receptor blockade alone, or in combination with inhibition of neutral endopeptidase (NEP), on the pharmacokinetics and metabolism of ANF in the rat. C-ANF-(11-15) infusion decreased apparent volume of distribution (Vss) and metabolic clearance rate (MCR) of administered 125I-ANF-(1-28) to one-third of their control values, whereas PHO alone was without effect on these parameters. In combination with C-ANF-(11-15), however, PHO further decreased MCR of 125I-ANF-(1-28) and increased plasma half time by more than threefold. High-performance liquid chromatography analysis revealed that C-ANF-(11-15) inhibited the delayed appearance of free 125I and [125I]monoiodotyrosine but had no effect on the small proportion of NEP metabolites in plasma. The combination of C-ANF-(11-15) and PHO further delayed the appearance of small metabolites, abolished the appearance of NEP metabolites, and markedly prolonged the permanence of intact 125I-ANF-(1-28) in plasma. The results demonstrate that C-ANF receptor blockade by C-ANF-(11-15) impairs clearance and metabolism of ANF, an effect which is synergistically potentiated by concomitant inhibition of NEP. C-ANF-(11-15) alone or in combination with NEP inhibitors may be a potentially useful therapeutic tool in the treatment of cardiovascular and renal diseases.

ANP-(7-23) stimulates a DHP-sensitive Ca2+ conductance and reduces cellular cAMP via a cGMP-independent mechanism.

Atrial natriuretic peptide (ANP) potently inhibits aldosterone secretion from the adrenal glomerulosa cell. In many tissues ANP action is associated with an increase in cellular guanosine 3',5'-cyclic monophosphate (cGMP) mediated through binding of the peptide to one of its receptors [ANP-A(R1)]. However, in the adrenal glomerulosa cell, the physiological significance of this rise in cGMP content has been contested. In an effort to determine whether non-cyclase-containing ANP receptors, such as ANP-C(R2), are linked to any of the events triggered by ANP binding, we utilized a truncated ANP analogue, ANP-(7-23), which at low doses exhibits selectivity for the ANP-C(R2) receptor. With the use of bovine adrenal glomerulosa cells, low concentrations (1 nM) of ANP-(7-23) failed to stimulate cGMP production, did not lower cytosolic calcium in the presence of low K+, and did not inhibit aldosterone secretion. At 1 nM, however, the analogue decreased cellular adenosine 3',5'-cyclic monophosphate content [8.27 +/- 0.51 vs. 6.74 +/- 0.09 (SE) pmol/10(6) cells; P less than 0.02] and, only in the presence of high extracellular [K+], increased cytosolic calcium. This ANP-induced rise in cytosolic calcium was abolished by the addition of a low dose (30 nM) of the dihydropyridine nitrendipine. ANP-(7-23) when utilized at a higher concentration (500 nM) lost its selectivity for the ANP-R2 receptor and increased cellular cGMP content (control, 0.27 +/- 0.02 vs. 500 nM ANP-(7-23), 0.448 +/- 0.02 pmol/10(6) cells; P less than 0.01). At 500 nM, ANP-(7-23) also inhibited aldosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of small C-ANF receptor ligands on plasma levels of atrial natriuretic factor, blood pressure, and renal function in the rat.

In this article, after a very brief review on ANF receptors, we report our study on the effects of small C-ANF receptor ligands in the rat. Two small ligands were synthesized: 2-naphthoxyacetyl-isonipecotyl-rANF11-15-NH2 (5 aa), containing 5 amino acids; and Ala7-rANF8-17-NH2 (10 aa), containing 10 amino acids from the ring structure of ANF1-28. After control periods, 5 aa or 10 aa were infused i.v. at a dose of 10 micrograms.min-1.kg-1 body weight for 70 min in anesthetized rats, followed by a 60-min recovery period. The 5 aa and 10 aa peptides significantly and reversibly increased plasma levels of endogenous immunoreactive ANF by 106 +/- 29 and 52 +/- 24 pg/mL, respectively. Infusion of the 5 aa peptide significantly decreased mean arterial blood pressure from 113 +/- 1 to 100 +/- 3 mmHg (1 mmHg = 133.32 Pa) and increased glomerular filtration rate from 1.6 +/- 0.2 to 2.3 +/- 0.2 mL/min, sodium excretion from 0.6 +/- 0.3 to 3.4 +/- 0.4 mumol/min, and potassium excretion from 0.5 +/- 0.2 to 1.2 +/- 0.2 mumol/min. Similar results were obtained with the 10 aa peptide. The effects of both peptides on blood pressure and sodium excretion persisted throughout the recovery period. The results confirm and extend previous observations showing that C-ANF receptors mediate the removal of ANF from the circulation. The shortening of the minimal peptide length necessary to bind to C-ANF receptors markedly enhances the possibility of developing orally active C-ANF receptor ligands for the treatment of cardiovascular and renal diseases.

Cellular mechanisms of the clearance function of type C receptors of atrial natriuretic factor.

Clearance (C) receptors of atrial natriuretic factor (ANF) have an important role of removing ANF from the circulation. In the present study we investigated the cellular mechanisms of this function. 125I-ANF1-28 specifically bound to C-ANF receptors in cultured bovine vascular smooth muscle cells is internalized by a temperature-dependent process at a rate of 5% occupied receptors/min at 37 degrees C. Internalized 125I-ANF1-28 is rapidly metabolized and released to the medium as [125I]monoiodotyrosine, a process that is reversibly inhibited by NH4Cl (10 mM). Retroendocytosis of receptor-ligand complex is detectable when intracellular accumulation of the ligand is maximized by previous incubation with NH4Cl. In the presence of saturating concentrations of 125I-ANF1-28 when cells are warmed from 4 to 37 degrees C, there is first a decrease and then an almost complete replenishment of surface C-ANF receptors, a phenomenon that is not altered by protein synthesis inhibition with cycloheximide. In the absence of temperature transition, the density of surface C-ANF receptors remains constant, and by 2 h of incubation the amount of ANF hydrolyzed far exceeds the total amount of C-ANF receptors. The total population of surface C-ANF receptors is internalized and recycled every hour, and these processes are constitutive since they also occur in the absence of ligand. Trypsinization and solubilization experiments further indicate that C-ANF receptors are rapidly internalized and recycled to the cell membrane. The results demonstrate that receptor-ligand internalization, extensive receptor recycling, and lysosomal hydrolysis of ANF are the cellular mechanisms by which C-ANF receptors exert their clearance function.

Hemodynamic effects of atrial natriuretic factor clearance receptor occupancy in conscious sheep.

The present study examined the hemodynamic actions of a non-guanylate cyclase linked or "clearance" atrial natriuretic factor (ANF) receptor ligand--des[Gln116Ser117Gly118Leu119Gly120] ANF 102-121 (C-ANF 4-23)--in conscious sheep. The effect of this peptide on the duration and potency of the hypotensive action of ANF (99-126) was also studied. C-ANF (4-23), infused at 400 micrograms/h for 2 h, reduced blood pressure, cardiac output and stroke volume, and increased total peripheral resistance slightly. These changes were similar to those previously observed with infusion of 20 micrograms/h ANF (99-126) in sheep. Endogenous ANF concentration increased from 28 +/- 13 to 85 +/- 18 pg/mL after 80 min infusion of C-ANF (4-23). The duration of hypotensive action from injection of ANF (99-126) was increased almost two-fold during infusion of C-ANF (4-23), however the hypotensive potency of ANF (99-126) was similar both prior to and during infusion of C-ANF (4-23). These studies support the concept of the metabolism of ANF via clearance receptors, suggesting that long-term hemodynamic actions of endogenous ANF may be achieved via prolonged blockade of these clearance receptors.

Isolation and functional expression of the human atrial natriuretic peptide clearance receptor cDNA.

The amino acid sequence of the human atrial natriuretic peptide clearance receptor (ANP C-receptor) was deduced from the nucleotide sequence of cDNA clones obtained from human placental and kidney cDNA libraries. The human sequence is highly homologous to the bovine C-receptor sequence already described, and the corresponding mRNA is expressed in human placenta, adult and fetal kidney and fetal heart. Upon transfection of this cDNA into mammalian cells, recombinant expression experiments revealed that the human ANP C-receptor has a high affinity for ANP (6 x 10(-9) M), similar to that observed for the receptor in other species. These data indicate that the human ANP C-receptor, previously characterized in other mammalian species, is highly conserved structurally and is expressed in various human tissues.

Human and canine gene homologs of porcine brain natriuretic peptide.

A cDNA clone encoding porcine brain natriuretic peptide (BNP) precursor was used to probe a canine genomic DNA library for homologous sequences. A unique clone was obtained (D1) which encoded a peptide homologous to porcine BNP. A 4.0 kB Hind III fragment of D1 containing the entire BNP precursor coding region was hybridized against blots of EcoRI digested human genomic DNA. A 7.0 kB hybridizing band was observed, which was subsequently subcloned and sequenced. This human clone, H1, also contained a sequence homologous to porcine BNP precursor. Transcription of the human BNP gene was confirmed by detection of BNP-specific sequences amplified from human atrial RNA by polymerase chain reaction. These results suggest that both BNP and ANP are present and function in a wide variety of mammalian species.

Expression and regulation of ANP receptor subtypes in rat renal glomeruli and papillae.

The expression and regulation of atrial natriuretic peptide (ANP) receptor subtypes were examined in rat renal glomeruli and papillae. In glomeruli, approximately two-thirds of ANP binding sites represented guanylate cyclase-uncoupled ANP clearance receptors (ANPc) with a molecular mass of 64 kDa under reducing conditions. The remainder of glomerular ANP binding sites represented guanylate cyclase-coupled ANP receptors (ANPGC) with a molecular mass of 130 kDa. In rat papillae, only the 130-kDa ANPGC was expressed. In rats adapted to a low-salt diet, saline loading or acute ANP infusion resulted in a decrease in ANPC density, a difference that was not detected when glomeruli were first acidwashed to remove endogenous ANP, indicating that apparent regulation of ANPC reflected prior occupancy by endogenous ANP. Densities of glomerular ANPC and ANPGC were similar in spontaneously hypertensive rats (SHR) compared with those of the Wistar-Kyoto (WKY) controls. However, elimination of prior receptor occupancy revealed a significantly greater expression of glomerular ANPC in SHR compared with WKY rats, without significant differences in the density of the glomerular or papillary ANPGC subtype. The failure of the ANPGC subtype to be regulated may account for our previously reported findings that dietary salt intake does not affect glomerular ANP-stimulated guanosine-3',5'-cyclic monophosphate accumulation despite apparent regulation of ANP receptor density. Whether the increased expression of the ANPC subtype in SHR represents a primary defect or results from induction of ANP clearance receptor expression remains to be determined.

Recombinant expression of a secreted form of the atrial natriuretic peptide clearance receptor.

A general structure for the atrial natriuretic peptide clearance receptor (ANP C-receptor) has been proposed based on hydropathicity analysis of the deduced amino acid sequence of this membrane protein (Fuller, F., Porter, J.G., Arfsten, A., Miller, J., Schilling, J., Scarborough, R.M., Lewicki, J.A., and Schenk, D.B. (1988) J. Biol. Chem. 263, 9395-9401). The ANP C-receptor is believed to possess a large amino-terminal extracellular domain (436 amino acids), a single hydrophobic transmembrane anchor (23 amino acids), and a short cytoplasmic tail (37 amino acids). As a means of testing the structure and proposed cellular orientation of this protein, we have employed the technique of in vitro mutagenesis to prepare a receptor mutant (anc-) lacking the transmembrane and cytoplasmic domains. Expression of this mutant in mammalian cells using a vaccinia virus vector results in secretion of a truncated soluble form of the ANP C-receptor which binds native ANP and synthetic ANP analogs with a specificity similar to that of the native ANP C-receptor. In contrast to the native ANP C-receptor that exists predominantly as a homodimer on the cell surface, the secreted receptor exists as a monomeric species. The results are consistent with the proposed structure of this receptor with the amino-terminal domain containing the ANP-binding site oriented extracellular to the plasma membrane. In addition, these data demonstrate that the receptor does not require association with the plasma membrane or its native dimeric configuration in order to bind ANP ligands with high affinity and specificity.

Cloning of a cDNA encoding porcine brain natriuretic peptide.

Complimentary DNA (cDNA) clones encoding porcine brain natriuretic peptide (BNP) were isolated from a porcine atrial cDNA library. The longest of the cDNA clones (1507 nucleotides) apparently originated from an unprocessed messenger RNA, since the nucleotide sequence encoding BNP-26 was interrupted by an intron of 554 nucleotides. A partial cDNA clone representing processed BNP mRNA was prepared by polymerase chain reaction. A comparison of the sequence of these two cDNAs reveals the presence of an additional intron within the sequence encoding the BNP precursor. The identification of these introns suggests that the BNP gene structure differs from the atrial natriuretic peptide gene in the location of intron 2. BNP mRNA encodes a propeptide of 131 amino acids, including a signal peptide domain (25 amino acids) and a prohormone domain (106 amino acids). Like atrial natriuretic peptide, the bioactive BNP sequence is localized at the carboxyl terminus of the prohormone. Although the carboxyl-terminal peptide sequences of porcine atrial natriuretic peptide and BNP are well conserved, there is relatively little homology within their propeptide regions.

Structure of recombinant human renin, a target for cardiovascular-active drugs, at 2.5 A resolution.

The x-ray crystal structure of recombinant human renin has been determined. Molecular dynamics techniques that included crystallographic data as a restraint were used to improve an initial model based on porcine pepsinogen. The present agreement factor for data from 8.0 to 2.5 angstroms (A) is 0.236. Some of the surface loops are poorly determined, and these disordered regions border a 30 A wide solvent channel. Comparison of renin with other aspartyl proteinases shows that, although the structural cores and active sites are highly conserved, surface residues, some of which are critical for specificity, vary greatly (up to 10A). Knowledge of the actual structure, as opposed to the use of models based on related enzymes, should facilitate the design of renin inhibitors.

Clearance function of type C receptors of atrial natriuretic factor in rats.

The overwhelming majority of atrial natriuretic factor (ANF) receptors in kidney and vascular tissues do not mediate any of the known functional effects of the hormone. To test whether these receptors (C-ANF receptors) function as clearance receptors for circulating ANF-(1-28), we determined the effects of C-ANF-(4-23) [des[Gln18Ser19Gly20Leu21Gly22]rANF-(3-23)-NH2], a specific ligand of C-ANF receptors, on the pharmacokinetics and hydrolysis of 125I-labeled ANF-(1-28) in anesthetized rats. Radioactivity in plasma was characterized by trichloroacetic acid solubility and high-pressure liquid chromatography. C-ANF-(4-23) (1 and 10 micrograms.min-1.kg body wt-1) led to marked dose-dependent increases in initial plasma concentration of administered 125I-ANF-(1-28) and decreases in its volume of distribution at steady state (Vss), metabolic clearance rate (MCR), and appearance of hydrolytic products ([125I]monoiodotyrosine and free 125I) in plasma (Pm). At the highest dose, C-ANF-(4-23) decreased Vss from 97 +/- 12 to 36 +/- 2 ml/100 g body wt, MCR from 50 +/- 4 to 12 +/- 1 ml.min-1.100 g body wt-1, and Pm from 54 +/- 8 to 11 +/- 2% of initial plasma 125I-ANF-(1-28). The data demonstrate that C-ANF receptors are mainly responsible for the very large volume of distribution and fast MCR of ANF in the rat. In this manner, C-ANF receptors are likely to play an important role in the homeostasis of circulating ANF.

Characterization of the atrial natriuretic peptide clearance receptor using a vaccinia virus expression vector.

A recombinant vaccinia virus has been used to direct the expression of the atrial natriuretic peptide clearance receptor (ANP C-receptor) in mammalian cell lines normally deficient in this protein. The recombinant receptor binds 125I-ANP-(102-126) in a specific and saturable manner and carboxyl-terminal truncated and internal-deleted ANP analogs bind to this site with high affinity. Following the covalent attachment of 125I-ANP-(102-126) to the recombinant ANP C-receptor, the protein exhibits an electrophoretic mobility identical to that of the native ANP C-receptor of cultured vascular cells. Expression of the ANP C-receptor in heterologous cells does not affect ANP-stimulated cyclic GMP accumulation, confirming previous reports that this novel ANP receptor subpopulation is not coupled to cyclic GMP metabolism. Furthermore, specific antisera, generated by inoculating rabbits with living recombinant virus, block 125I-ANP binding to the ANP C-receptor but do not inhibit ANP stimulation of cyclic GMP, supporting the existence of two receptor subpopulations that are functionally and immunologically distinct.

D-amino acid-substituted atrial natriuretic peptide analogs reveal novel receptor recognition requirements.

The introduction of D-amino acid residues into peptide hormones has been traditionally utilized in structure-activity studies to probe the conformational requirements of ligand-receptor interactions. A study was undertaken to examine the effect of D-amino acid substitutions into the atrial natriuretic peptide molecule on interactions with distinct subpopulations of specific membrane-associated receptors of bovine aortic smooth muscle cells. Competitive binding analysis revealed that each of 15 synthetic D-amino acid-substituted analogs showed comparable affinities for C-ANP receptors, a class of specific receptors which have been proposed to mediate the sequestration and metabolic clearance of ANP. The relative affinities of all 15 analogs did not differ more than 10-fold. In contrast, the interaction of the ANP analogs with a second receptor pool (B-ANP receptors), which is coupled to the stimulation of particulate guanylate cyclase, varied over a 1000-fold range of potency consistent with expectations for a receptor that displays rigorous conformational specificity. The indiscriminant selectivity of C-ANP receptors for D-amino acid-substituted ANP analogs is unprecedented for hormone receptors involved in biological signal transduction. These results, when coupled with the inability to correlate any direct in vitro biological effect associated with C-ANP receptor occupancy supports the hypothesis that the C-ANP receptor protein is a novel transport protein involved in the metabolic clearance of ANP.

Semi-preparative purification of recombinant human renin and prorenin.

Chinese hamster ovary (CHO) cells, transfected with a vector containing cDNA coding for preprorenin, have been shown to secrete authentic prorenin into the culture supernatant. Purification of the expressed prorenin and purification of active renin, generated by solid-phase trypsin treatment of the conditioned media, have been achieved by conventional chromatographic methods. Scale-up of the initial steps of these procedures is described, including the use of radial-flow columns and automation with fast protein liquid chromatography valves and pumps. This semi-preparative scheme has allowed hundreds of milligrams of both proteins to be isolated.

Atrial natriuretic peptide clearance receptor. Complete sequence and functional expression of cDNA clones.

The major class of atrial natriuretic peptide (ANP) receptors was isolated from cultured vascular smooth muscle cells, and a partial amino acid sequence was obtained. This allowed the isolation of cDNA clones from which the entire amino acid sequence was established. The smooth muscle cell ANP receptor appears to be synthesized as a 537-amino acid precursor with an N-terminal membrane translocation signal. The mature form consists of 496 amino acids with a single potential transmembrane domain predicting a 37-amino acid cytoplasmic domain and a large, acidic, extracellular domain low in cysteine and probably containing attached carbohydrate. The receptor is therefore similar in structure to the growth factor receptors but notably lacks repetitive cysteine-rich domains and has a relatively small intracellular domain. Expression of the cloned receptor in Xenopus oocytes elicited high affinity, membrane-associated binding sites for ANP and for truncated and internally deleted analogs of ANP. These results reflect the ligand binding specificity found for the major class of ANP receptors on smooth muscle cells and thus provide additional evidence that two distinct ANP receptors exist since ANP receptor-coupled guanylate cyclase activity exhibits a very different ANP analog specificity.